Assaying Protein Kinase Activity with Radiolabeled ATP

Summary

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([γ-³²P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Abstract

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-³²P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Introduction

Protein kinases are sophisticated enzymes critical for the transmission of cellular signals into appropriate responses¹. Given their roles in maintaining homeostasis and the prevention or promotion of disease states², biochemical methods for assessing kinase activity continue to be powerful tools for delineating the particulars of eukaryotic signaling³. Although strategies utilizing phospho-specific antibodies have been highly informative in terms of measuring the effects of different treatment conditions on cell signaling status⁴, a kinase assay allows for measuring the ef....

Protocol

1. Kinase Purification Resources and General Immunoprecipitation Pipeline

NOTE: Kinases for use with this assay may be sourced from immunoprecipitates of cultured cells or by recombinant means such as affinity-tagged purification⁶. Below is a general protocol for flag-tagged immunoprecipitation that may have to be modified depending on the kinase of interest. Everything should be kept on ice when possible.

1. Add 2 µL of 1 mg/mL antibody to 200 µL of cell lysates (usually ~1 mg/mL total protein concentration) and incubate at 4 °C for 1 h while rocking.
2. Wash protein A sepharos....

Results

WNK1 IP kinase assays

Myc-tagged WNK1 was transfected into HEK293 cells and immunoprecipitated with an anti-Myc antibody¹¹. The immunoprecipitate displayed kinase activity towards the model substrate MBP as well as toward itself (Figure 1A). WNK1 mutants were then tested for kinase activity towards MBP by the same method, this time employing GST-tagged constructs (Figure 1B).......

Discussion

Kinases are a diverse family of proteins that have evolved extensive functionality in numerous contexts, and kinase assays have been incredibly useful in studying several signaling proteins and have greatly contributed to our current understanding of cellular communication. Notably, the same basic assay was used in characterizing two disparate kinases despite major differences in structure and activity. WNK1 kinase contains an atypical catalytic pocket where the critical lysine has shifted to a unique position and is kno.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protein A Sepharose CL-4B	GE Healthcare Life Sciences	17-0963-03
Radiolabeled [γ-32P] ATP	Perkin Elmer	NEG035C010MC
Laemmli Buffer	Bio-Rad	#1610737
Radioactive shielding	Research Products International	BR-006
R-250 dye (Coomassie)	Thermo Fisher	20278
Whatman Grade 3 qualitative filter paper	Whatman	1003-917
Slab gel vacuum dryer	Bio-Rad	Model 583 Gel Dryer #1651745
Autorad marker	Agilent Technologies	420201
Phosphorescent ruler	Sigma Aldrich	R8133
Phosphorescent dots	Sigma Aldrich	L5149
Geiger counter	Ludlum	Model 3 with 44-9 detector
BioMax Cassette 8 x 10"	Carestream Health	107 2263
BioMax MS Film 8" x 10"	Carestream Health	829 4985
BioMax MS Intensifying Screen 8" x 10"	Carestream Health	851 8706
Medical/X-ray film processor	Konica Minolta	SRX-101A
Scintillation vials	Research Products International	125500
Scintillation fluid	MP Biomedicals	188245305
Beckman LS 6500 Scintillation counter	Beckman	LS 6500